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Characterization and Quantification of Lyophilized Product Appearance
and Structure
Dr Kevin R. Ward B.Sc. Ph.D. MRSC
Director of Research & Development Biopharma Technology Ltd.
Winchester SO23 0LD, UK Tel: +44 (0)1962 841092
E-mail: [email protected] Web: www.btl-solutions.net
ISL-FD Conference, Bologna, Italy, March 2012
Synopsis of Presentation
• Subjectivity of product appearance assessment
– Macroscopic features of lyophiles – Descriptors that might be used – Microscopic features
• What other features related to appearance and structure can be quantified?
– Porosity
– Specific surface area
– Density variations within a cake
– Stress / strain testing for mechanical properties
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Acknowledgements
Dr Daryl Williams Sharmila Devi
My team at BTL:
• Isobel Cook
• Tom Peacock
• Mervyn Middleton
• Nick White
for sponsoring Imperial College studentship
Dr Andrew Ingham Edmond Ekenlebie
Lyophile Characterization
• Appearance
• Residual moisture (KF, LoD, NIR, FMS…)
• Container seal integrity (pressure)
• Oxygen levels in vials / ampoules
• Molecular integrity assays
• Activity and Stability assays
• Crystal / polymorphic forms / hydrates
• Thermal properties – Tg, relaxation times
• Reconstitution properties
Appearance seems to be the only parameter that is not usually quantified!
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Product Appearance
• Difficult to quantify by eyesight alone!
• We can tell total collapse and partial collapse from something that is not collapsed…
Product Appearance
• Or a product that has undergone severe eutectic melting…
• Or a friable one that became powdery…
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Product Appearance
• Or where there almost seem to be several different products in one vial…!
Disc on top (due to crust formation or early collapse)
Powder underneath
Product Appearance
• But of the ones that appear to look “good”, there are still differences…
Uniform; adhered to vial Uniform; not adhered to vial Does the peak matter?!
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Some Features / Adjectives
Texture: rough, smooth, porous, “grainy”…
Surface features (peak,
sheen, skin, crust…)
Adhesion to vial
Cake height (vs. fill height)
Shape
Cohesive or powdery?
Colour
(or shade of white?!)Uniformity (homogeneous / heterogeneous?)
Collapsed or melted /
…but what about the microscopic differences we can’t see?
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Electron microscopy
SEM, TEM
• Can be used to look at porosity, microstructure, possible microcollapse
But remember, the image may depend on sampling technique:
• Is the sample representative of the whole cake?
• Has the sampling process changed it in any way?
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SEM analysis and annealing
For mannitol + sucrose mixture
Annealed sample:
Mannitol crystallised and/or larger ice crystals, giving
‘rougher’ structure
Non-annealed sample:
Mannitol amorphous and/or ice crystals smaller, giving ‘smoother’
structure
However, this method is still not quantitative!
Methods of quantifying appearance and structure
• Gas adsorption methods
– Specific surface area – Mean pore diameter
• Micro-CT scanning
– Porosity
– Heterogeneity
• Stress / strain testing
– Mechanical properties
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Surface Area & Porosity
by gas adsorption methods
• N2 sorption based on Brunauer-Emmett-Teller
(BET) and Barrett-Joyner-Halenda (BJH) / Kelvin equations
• BET and BJH equations allow determination of specific surface area and pore size distribution
• We carried out a simple study for mannitol:
– Nitrogen adsorption and desorption isotherms were measured at -195.8°C, using an ASAP Tristar 3000 (Micromeritics Instrument Corporation, USA)
volumetric adsorption system
• Similar isotherms but hysteresis is wider for the LN2 quench‐cooled mannitol
• This indicates that the energy needed for evaporation from the pores is distinctly different from the energy associated with condensation within it
• This in turn implies that desorption (evaporation) is inhibited due to constriction, thereby suggesting pores are smaller
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BET adsorption analysis
For surface area of cake
Specific surface area, m2 /gram
0 1 2 3 4 5 6 7 8
Slow cool Fast cool LN2 cool
SA ads SA des SA BET
Data for mannitol lyophilized using different initial cooling rates (BTL / ICL)
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BJH adsorption analysis
For mean pore diameter
0 100 200 300 400 500 600
Slow cool Fast cool LN2 cool
ads pore diam des pore diam
Mean pore diameter, Angstroms
Data for mannitol lyophilized using different initial cooling rates (BTL / ICL)
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Structural assessment:
Micro-CT scanning (tomography)
Dr Andrew Parker (Molecular Profiles) gave an excellent presentation on this technique at
Visiongain’s 2010 Lyo conference in London
Micro-CT scanning enables a 3D picture to be built up of a freeze-dried product, showing pore
structure and cake uniformity
Ref: “Determination of the influence of primary drying rates on the microscale structural attributes and physicochemical properties of
protein containing lyophilized products”, A. Parker et al, J. Pharm. Sci.
(published online 28/6/10). DOI: 10.1002/jps22185
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Three-dimensional cross-sectional X-ray CT into packed powders
Scale bar: 1.25 mm Freeze-dried mannitol powder Fluid bed dried mannitol powder
Images courtesy of Dr Andrew Ingham & Edmond Ekenlebie, Aston University, UK.
Taken from their publication “Short Cycle Times for Cost-Efficient Processing in Lyophilized Formulations”, American Pharmaceutical Review (2011)
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Mechanical Properties:
Stress / strain measurement
• In collaboration with Imperial College London, we are currently developing a miniature load cell to measure stress and strain in a lyophilized
cake while it is still in the vial
• Measuring the stress (σ) and strain (ε) can also give us elasticity, Young’s modulus (E):
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• Stable Microsystems Texture analyser and Lloyds EZ50.
• Loaded at speed of 1mm/sec, tested at 0.01mm/sec to a depth of 3mm (start at 1g force)
• Samples tested in the vial
• Samples: Fast, slow and LN quench cooled mannitol
Stress / Strain Testing
AIM:
To compare the
strength of the cakes
•Compression testing
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Data for mannitol
E modulus (N/m2) Mean Fast Cooled
60863
45428.5 29994
LN2 Quench Cooled
12213
8692 5171
Slow Cooled
3303
2328 1353
• Fast cooled mannitol strongest, slow cooled mannitol has lowest strength
• Could be due to morphology or fundamental material properties
• Morphology :
• Slow cooled has larger pores therefore the support may not be strong
• From the SEM pictures, small holes are visible in the LN2‐ and slow‐ cooled samples; holes are the weakest points for the cakes to crack/break easily
• Material property: Strength/physical stability of the polymorphic form but not clear
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Summary
• Subjectivity of product appearance assessment
– Macroscopic features of lyophiles – Descriptors that might be used – Microscopic features
• Other features related to appearance and structure that can be quantified
– Porosity
– Specific surface area
– Density variations within a cake
– Stress / strain testing for mechanical properties
Biopharma House, Winnall Valley Road, Winchester SO23 0LD, UK Tel: +44 (0)1962 841092 Web: www.btl-solutions.net
